This application proposes the study of the rapid regulation of the receptor for thiazide-type diuretics. It eminates from our unexpected finding of the unprecedented phenomenon of rapid regulation of this diuretic binding site. Specifically, the binding of 3H-metolazone to the receptor for thiazide-type diuretics in kidney is apparently i) up to 90% "down- regulated" or "unmasked" by 10 minutes of reperfusion in vivo or 10 minutes of re-oxygenation in vitro. Our discovery of this phenomenon is certainly the first description of such a finding for any diuretic drug and it may be the first description ever of such a rapid and complete regulation of a drug or ligand binding site in mammalian system. We outline studies under three discrete aims, which are: i) To describe fully the in vivo and in vitro time course and the in vitro conditions for the study of this phenomenon of raped regulation of the binding site for 3H-metolazone. ii) To use optimized incubation conditions for the in vitro detection and study of the cellular/metabolic pathways that are involved in this regulation. Many of these pathways fall under the rubric of "second messenger systems". The isolated perfused kidney will be used, if appropriate. iii) To establish a cell line, transformed in vitro, from primary cultures of renal distal nephron cells. Microdissected distal nephron cells will be infected with a retroviral construct containing the adenoviral oncogenes Ela (12s or 13s) and a dominant neomycin-resistant gene. When incorporated into the genome, the former will "immortalize" or transform the cell and the latter will enable selection of cells that have incorporated the construct. Clones will be tested for the ability to bid 3h-metolazone and positive clones expanded. Such a cell line should enable future study of the phenomenon of rapid regulation of binding of 3H-metolazone in parallel with the ion transport function of the thiazide-sensitive transporter.